Density and Correlation functions of vortex and saddle points in open billiard systems

We present microwave measurements for the density and spatial correlation of current critical points in an open billiard system, and compare them with the predictions of the Random Wave Model (RWM). In particular, due to a novel improvement of the experimental set-up, we determine experimentally the spatial correlation of saddle points of the current field. An asymptotic expression for the vortex-saddle and saddle-saddle correlation functions based on the RWM is derived, with experiment and theory agreeing well. We also derive an expression for the density of saddle points in the presence of a straight boundary with general mixed boundary conditions in the RWM, and compare with experimental measurements of the vortex and saddle density in the vicinity of a straight wall satisfying Dirichlet conditions.Comment: submitted to Physical Review

Classical wave experiments on chaotic scattering

We review recent research on the transport properties of classical waves through chaotic systems with special emphasis on microwaves and sound waves. Inasmuch as these experiments use antennas or transducers to couple waves into or out of the systems, scattering theory has to be applied for a quantitative interpretation of the measurements. Most experiments concentrate on tests of predictions from random matrix theory and the random plane wave approximation. In all studied examples a quantitative agreement between experiment and theory is achieved. To this end it is necessary, however, to take absorption and imperfect coupling into account, concepts that were ignored in most previous theoretical investigations. Classical phase space signatures of scattering are being examined in a small number of experiments.Comment: 33 pages, 13 figures; invited review for the Special Issue of J. Phys. A: Math. Gen. on "Trends in Quantum Chaotic Scattering

Cytochemical techniques and energy-filtering transmission electron microscopy applied to the study of parasitic protozoa

The study of parasitic protozoa plays a major role in cell biology, biochemistry and molecular biology. Numerous cytochemical techniques have been developed in order to unequivocally identify the nature of subcellular compartments. Enzyme and immuno-cytochemistry allow the detection of, respectively, enzymatic activity products and antigens in particular sites within the cell. Energy-filtering transmission electron microscopy permits the detection of specific elements within such compartments. These approaches are particularly useful for studies employing antimicrobial agents where cellular compartments may be destroyed or remarkably altered and thus hardly identified by standard methods of observation. In this regard cytochemical and spectroscopic techniques provide valuable data allowing the determination of the mechanisms of action of such compounds

The Mechanisms of Codon Reassignments in Mitochondrial Genetic Codes

Many cases of non-standard genetic codes are known in mitochondrial genomes. We carry out analysis of phylogeny and codon usage of organisms for which the complete mitochondrial genome is available, and we determine the most likely mechanism for codon reassignment in each case. Reassignment events can be classified according to the gain-loss framework. The gain represents the appearance of a new tRNA for the reassigned codon or the change of an existing tRNA such that it gains the ability to pair with the codon. The loss represents the deletion of a tRNA or the change in a tRNA so that it no longer translates the codon. One possible mechanism is Codon Disappearance, where the codon disappears from the genome prior to the gain and loss events. In the alternative mechanisms the codon does not disappear. In the Unassigned Codon mechanism, the loss occurs first, whereas in the Ambiguous Intermediate mechanism, the gain occurs first. Codon usage analysis gives clear evidence of cases where the codon disappeared at the point of the reassignment and also cases where it did not disappear. Codon disappearance is the probable explanation for stop to sense reassignments and a small number of reassignments of sense codons. However, the majority of sense to sense reassignments cannot be explained by codon disappearance. In the latter cases, by analysis of the presence or absence of tRNAs in the genome and of the changes in tRNA sequences, it is sometimes possible to distinguish between the Unassigned Codon and Ambiguous Intermediate mechanisms. We emphasize that not all reassignments follow the same scenario and that it is necessary to consider the details of each case carefully.Comment: 53 pages (45 pages, including 4 figures + 8 pages of supplementary information). To appear in J.Mol.Evo

Controlled assembly of SNAP-PNA-fluorophore systems on DNA templates to produce fluorescence resonance energy transfer

The SNAP protein is a widely used self-labeling tag that can be used for tracking protein localization and trafficking in living systems. A model system providing controlled alignment of SNAP-tag units can provide a new way to study clustering of fusion proteins. In this work, fluorescent SNAP-PNA conjugates were controllably assembled on DNA frameworks forming dimers, trimers, and tetramers. Modification of peptide nucleic acid (PNA) with the O6-benzyl guanine (BG) group allowed the generation of site-selective covalent links between PNA and the SNAP protein. The modified BG-PNAs were labeled with fluorescent Atto dyes and subsequently chemo-selectively conjugated to SNAP protein. Efficient assembly into dimer and oligomer forms was verified via size exclusion chromatography (SEC), electrophoresis (SDS-PAGE), and fluorescence spectroscopy. DNA directed assembly of homo- and hetero-dimers of SNAP-PNA constructs induced homo- and hetero-FRET, respectively. Longer DNA scaffolds controllably aligned similar fluorescent SNAP-PNA constructs into higher oligomers exhibiting homo-FRET. The combined SEC and homo-FRET studies indicated the 1:1 and saturated assemblies of SNAP-PNA-fluorophore:DNA formed preferentially in this system. This suggested a kinetic/stoichiometric model of assembly rather than binomially distributed products. These BG-PNA-fluorophore building blocks allow facile introduction of fluorophores and/or assembly directing moieties onto any protein containing SNAP. Template directed assembly of PNA modified SNAP proteins may be used to investigate clustering behavior both with and without fluorescent labels which may find use in the study of assembly processes in cells

Shale gas production: potential versus actual greenhouse gas emissions

Estimates of greenhouse gas (GHG) emissions from shale gas production and use are controversial. Here we assess the level of GHG emissions from shale gas well hydraulic fracturing operations in the United States during 2010. Data from each of the approximately 4000 horizontal shale gas wells brought online that year are used to show that about 900 Gg CH[subscript 4] of potential fugitive emissions were generated by these operations, or 228 Mg CH[subscript 4] per well—a figure inappropriately used in analyses of the GHG impact of shale gas. In fact, along with simply venting gas produced during the completion of shale gas wells, two additional techniques are widely used to handle these potential emissions: gas flaring and reduced emission 'green' completions. The use of flaring and reduced emission completions reduce the levels of actual fugitive emissions from shale well completion operations to about 216 Gg CH[subscript 4], or 50 Mg CH[subscript 4] per well, a release substantially lower than several widely quoted estimates. Although fugitive emissions from the overall natural gas sector are a proper concern, it is incorrect to suggest that shale gas-related hydraulic fracturing has substantially altered the overall GHG intensity of natural gas production

Structural bases for the interaction of frataxin with the central components of iron–sulphur cluster assembly

Reduced levels of frataxin, an essential protein of as yet unknown function, are responsible for causing the neurodegenerative pathology Friedreich's ataxia. Independent reports have linked frataxin to iron–sulphur cluster assembly through interactions with the two central components of this machinery: desulphurase Nfs1/IscS and the scaffold protein Isu/IscU. In this study, we use a combination of biophysical methods to define the structural bases of the interaction of CyaY (the bacterial orthologue of frataxin) with the IscS/IscU complex. We show that CyaY binds IscS as a monomer in a pocket between the active site and the IscS dimer interface. Recognition does not require iron and occurs through electrostatic interactions of complementary charged residues. Mutations at the complex interface affect the rates of enzymatic cluster formation. CyaY binding strengthens the affinity of the IscS/IscU complex. Our data suggest a new paradigm for understanding the role of frataxin as a regulator of IscS functions

Solution Structure of the Iron−Sulfur Cluster Cochaperone HscB and Its Binding Surface for the Iron−Sulfur Assembly Scaffold Protein IscU†‡

ABSTRACT: The interaction between IscU and HscB is critical for successful assembly of iron-sulfur clusters. NMR experiments were performed on HscB to investigate which of its residues might be part of the IscU binding surface. Residual dipolar couplings ( 1 DHN and 1 DCRHR) indicated that the crystal structure of HscB [Cupp-Vickery, J. R., and Vickery, L. E. (2000) Crystal structure of Hsc20, a J-type cochaperone from Escherichia coli, J. Mol. Biol. 304, 835-845] faithfully represents its solution state. NMR relaxation rates ( 15 N R1, R2) and 1 H- 15 N heteronuclear NOE values indicated that HscB is rigid along its entire backbone except for three short regions which exhibit flexibility on a fast time scale. Changes in the NMR spectrum of HscB upon addition of IscU mapped to the J-domain/C-domain interface, the interdomain linker, and the C-domain. Sequence conservation is low in the interface and in the linker, and NMR changes observed for these residues likely result from indirect effects of IscU binding. NMR changes observed in the conserved patch of residues in the C-domain (L92, M93, L96, E97, E100, E104, and F153) were suggestive of a direct interaction with IscU. To test this, we replaced several of these residues with alanine and assayed for the ability of HscB to interact with IscU and to stimulate HscA ATPase activity. HscB(L92A,M93A,F153A) and HscB(E97A,E100A,E104A) both showed decreased binding affinity for IscU; the (L92A,M93A,F153A) substitution also strongly perturbed the allosteric interactio